Grants

NSF

Microbial energy and mass transfers have profound ecological and biogeochemical consequences, yet the controls directing microbial metabolic trade-offs remain poorly understood. The proposed project will study metabolic shifts in energy and mass transfers by microorganisms living through some of the earliest-evolving metabolisms known on Earth. By focusing efforts towards microbes driving primary production anaerobically with molecular hydrogen — through a metabolic process known as chemosynthesis — the project will test whether metabolic trade-offs can be predicted as a function of (i) growth temperature and/or (i) the amount of free energy associated with anaerobic respiration. State-of-the-art continuous cultivation techniques will be applied to compare changes in respiration rates, cell yields, and cell doubling times within and among chemosynthetic microbes under steady-state conditions. These experiments are key for integrating parameters describing microbial metabolic changes into mathematical and/or thermodynamic models of microbial growth and its biogeochemical consequence. Overall, this work will transform understanding of the underlying metabolic behavior of unicellular life at the base of the Universal Tree of Life and at the base of dark ecosystems. The objective of the proposed research is to determine whether microbial metabolic rate/yield trade-offs of hydrogenotrophic NO3--, Fe3+- and CO2-reducing microorganisms exhibit predictable adaptations to temperature and to the oxidation-reduction (redox) chemistry of catabolism during chemosynthesis. Specifically, we will test the hypotheses that (i) microbial metabolic rate-to-yield ratios will increase for all proposed microbial processes when at higher temperatures and that (ii) microbes relying on generally less exergonic catabolic redox reactions will systematically display higher anabolic efficiencies than those relying on more exergonic catabolic redox reactions. To test these hypotheses, the project will employ experimental microbiological and geochemical approaches to compare microbial respiration rates, cell (and total biomass) yields, and cell doubling times of different anaerobic chemosynthetic microorganisms during batch and continuous growth. The resulting metabolic and environmental data will demonstrate how microbial metabolic behaviors are shaped by their abiotic environments. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIDCD - National Institute on Deafness and Other Communication Disorders

Project Summary Hundreds of taste buds (TBs) are located in the epithelial trenches of the circumvallate taste papilla (CVP) at the posterior midline of the tongue. Each TB contains taste receptor cells (TRCs) that transduce taste information to the brain and are continually replaced from stem/progenitor cells outside of TBs. Cancer patients undergoing chemo- or radiotherapy often experience dysgeusia, or taste dysfunction, likely due to perturbation to TRC renewal. Patients experiencing dysgeusia have increased risk of depression, malnutrition, and poor treatment outcomes. Therefore, understanding the mechanisms regulating maintenance of taste epithelium and TRC renewal will inform development of treatments to prevent or restore taste loss in these patients. CVP homeostasis occurs through proliferation and differentiation of progenitor cells that generate both TRCs and the surrounding non-taste epithelium. Our lab has recently identified new, long-term SOX9+ progenitors that, as a population, generate TRCs and non-taste epithelium (NTE); however, the potential of individual SOX9+ progenitors is unexplored. SOX9+ progenitors are located distant from taste buds, in the epithelial junction linking CVP trenches to the ducts of the underlying von Ebner's minor salivary glands (VEG). Each CVP contains a dozen or more junctions containing SOX9+ cells, suggesting that multiple independent progenitor reservoirs contribute to taste epithelium. Additionally, in contrast to the highly proliferative CVP, the junction contains few proliferative cells, indicating SOX9+ cells are slow cycling. These findings lead to my hypothesis that individual SOX9+ progenitors from multiple junctions produce progeny that move into local CVP trench epithelium, become highly proliferative, and differentiate into TRCs and NTE. Thus, in Aim 1, I will use sparse SOX9 lineage trace, whole-tissue clearing, and EdU birth dating, to determine the lineage and proliferative potential of individual progenitors from multiple junctions. Our previous work has focused on SOX9+ progenitor contribution in adult homeostasis, but if and when these taste stem cells are established embryonically is unknown. Embryonic immunostaining reveals SOX9+ cells are present during initial formation of the CVP and have overlapping expression with Ptch, the Shh signaling receptor. Shh is necessary for anterior tongue taste bud development, and proper CVP trench invagination in the posterior tongue. These findings lead to my second hypothesis that embryonic Shh signaling is required to establish adult SOX9+ taste progenitors. In Aim 2, I will use embryonic lineage tracing and genetic deletion of Shh signaling in SOX9+ cells to determine when SOX9+ cells are established, begin contributing to TBs, and if Shh signaling is required in these processes. Overall, this work may lead to approaches to leverage concentrated pools of taste stem cells to restore taste function in patients.

NSF

This project explores how macrodomain proteins have evolved to balance their different multiple biochemical activities. Macrodomains are ancient enzymes that bind to and remove ADP-ribose, an important post-translation modification that is critical in several cellular stress responses, including DNA damage, ER stress, and virus infection. These enzymes are conserved through all domains of life, including archaea, bacteria, eukaryotes, and viruses, indicating that they are critical for multiple cellular processes. However, the function of macrodomain enzymes in cell biology and microbiology are just now being uncovered. Furthermore, recent research indicates that macrodomains have evolved to properly balance their biochemical functions depending on if they are expressed from a virus, a bacteria, or from a eukaryotic cell. This project will evaluate how viral macrodomains have evolved to develop the ideal biochemical properties that allow them function in the context of a virus infection. This project also has strong educational and community outreach components. Most notably, students at all training levels will participate in this project and will learn how to evaluate the evolution of proteins through workshops in phylogenetics. This project will provide new insights into the fundamental biology of macrodomain enzymes and could lead to new insights into antiviral drug-development. This project aims to define how the coronavirus macrodomain has evolved to best function in the context of a virus infection using the mouse coronavirus, murine hepatitis virus (MHV), as a model. The use of MHV, which is unable to infect humans, for creating macrodomain mutations eliminates the potential for gain-of-function research. Research over the last decade has demonstrated that the coronavirus macrodomain blocks innate immune responses, is critical for viral pathogenesis, and is a potential drug target. Furthermore, the macrodomain uses both its ADP-ribose binding and hydrolysis activities to promote virus replication, and they must be balanced for optimal replication. The PI will use recombinant proteins and a panel of recombinant viruses, developed using a bacterial artificial chromosome based reverse genetic system, to better understand how highly conserved amino acids and selective pressure induced mutations impact Mac1 biochemical activities, virus replication, and pathogenesis using well-established assays and model systems. Additionally, macrodomains from across the evolutionary spectrum will be expressed in the context of MHV infection to define the evolutionary limits of macrodomain divergence that is tolerated in MHV. This work will provide a deeper understanding of how macrodomains have evolved to counter various ADP-ribose dependent antiviral responses. This project is jointly funded by the Genetic Mechanisms Program and the Division of Molecular and Cellular Biosciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Pneumonic plague is the deadliest form of disease caused by Yersinia pestis. Disease manifests as a rapidly progressing and highly lethal necrotic pneumonia that is typically fatal within seven days. If antibiotic treatment is not administered within 24 hours after the onset of symptoms, pneumonic plague is 100% fatal. The lethality of plague is due to the abrupt onset of a hyper-inflammatory response that compromises pulmonary function. Deletion of the Y. pestis inner membrane protein YbtX results in decreased neutrophil infiltration into the airways and decreased expression of select pro-inflammatory cytokines/chemokines during the later stages of pneumonic plague. YbtX is part of a zinc acquisition system by which the Y. pestis siderophore Yersiniabactin (Ybt) competes with host protein calprotectin to scavenges bioavailable zinc. YbtX acts as an inner membrane transporter that facilitates transport of Ybt-Zn into the bacterial cytosol. Calprotectin is also known to be a driver of host inflammatory responses. We hypothesize that YbtX-mediated zinc import contributes to the onset inflammation in the lung by activating calprotectin-dependent inflammatory responses. To test this, we will characterize how zinc depletion impacts inflammation during infection in the absence of YbtX. Further, we will alter the balance of host zinc/calprotectin in a murine intranasal infection model of pneumonic plague and characterize how this balance affects pulmonary inflammation. This work will determine how zinc and host calprotectin impact host inflammatory responses during pneumonic plague.

National Park Service

United State Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This posting is to provide public notice of NPS' intention to fund the following project activities without full and open competition: Task Agreement P14AC01558 under Cooperative Agreement H8W07110001 with the University of Washington, a partner under the Pacific Northwest Cooperative Ecosystem Studies Unit, for the project titled, "Determining if Managed Wildfires and Prescribed Fires Conserve Critical Habitat Structure for Pacific Fishers in the Southern Sierra Nevada."

NIA - National Institute on Aging

Project Summary Abstract Alzheimer disease (AD) is the most common form of dementia that has no cure and few therapeutic options, and the strongest risk factor for developing AD dementia is aging, an unavoidable event. T cells are greatly affected during aging as they become comprised of less naïve/resting cells and more terminal differentiated/senescent cells that can be cytotoxic and produce high amounts of pro-inflammatory cytokines. Inflammation is known to be an early event in AD with innate microglial activation and an augmented cytokine production in the CNS and increases in frequency of circulating Th1, TH17 and Th9 inflammatory T-cells. Functioning in distinct contrast to these inflammatory T cells, are regulatory T cells (Tregs) which are a small, usually stable, lineage of inhibitory CD4 T cells that expresses the FoxP3 transcription factor and is essential for maintaining healthy homeostasis and tolerance. Tregs appear to be involved in AD pathology as most reports have found them to be at low frequency in the AD patient circulation, while mouse AD models demonstrate that Tregs can affect the disease as the adoptive transfer of Tregs slows disease progression. The ability of Tregs to down modulate inflammation makes them central to immune resolution and inhibition of autoimmunity. But Tregs have additional far-reaching activities as they not only can inhibit the activation of microglia and other immune lineages (B, T, monocyte), but also can initiate tissue repair and recovery. Tregs have been shown to be neuroprotective in mouse models for brain hemorrhage and for the neurodegenerative diseases Parkinson’s, ALS, and AD. Excitingly, the administration of wild type Tregs into the APPPS1 AD mouse model was found to slow disease and also induce recovery of cognition. Yet, only one group has actually isolated and performed the in vitro functional assays needed to determine if AD patient-derived Tregs are functionally competent or if their reduced activity would represent a potential therapeutic target. Unfortunately, this study used suboptimal Treg isolation methods, combined functionally-distinct Treg subsets into a single population, and analyzed only one parameter to assess Treg function – suppression of target cell proliferation. Thus, it is crucial that a more comprehensive study be performed to reveal the breadth of potential dysfunction of Tregs in AD-dementia and expose dysfunction-inducing mechanisms. As our lab routinely examines human Treg function in disease, we have begun to study AD-Tregs using a high stringency isolation and multi-parameter functional assay approach that separately interrogates functionally distinct subsets of Tregs isolated from pairs of matched AD-dementia vs HC PBMCs. Our overarching goal is to uncover if and why Tregs show dysfunction in AD. Illuminating the mechanisms of Treg deficiency in AD will contribute to future development of Treg-directed therapies for AD, where optimal Tregs may slow progression, induce tissue repair and restore cognition.

NCI - National Cancer Institute

PROJECT SUMMARY Colorectal Cancer (CRC) is a significant burden on human health and in need of better diagnostics and treatments. Microbes have been implicated in ~20% of all human cancers, and recent studies have shown strong associations of CRC with bacteria residing in the gut. The gut microbiota has well-established roles in modulating intestinal immune responses that can exacerbate or alleviate inflammation, however, the ways in which members of the gut microbiota modulate pro or anti-tumor immune responses remain largely unknown. Colonic tumors are infiltrated by surrounding immune cells and disease outcome is heavily influenced by which immune cells are recruited to tumors and their roles within the tumor microenvironment (TME), therefore, understanding the mechanisms of how gut bacteria can modulate those infiltrating immune cells is critical in designing new and more improved diagnostics and interventions. In my work investigating human microbiotas from CRC patients, I discovered a human gut commensal, Bacteroides uniformis, protects against disease by enhancing anti-tumor immunity. This protection is dependent on Natural Killer (NK) cells and is effective in multiple pre-clinical mouse tumor models. I found that B. uniformis directly activates NK cells, and this effect is independent of adaptive immunity. However, the bacterial factors recognized by NKs are still unknown, as are the dynamics within the NK cells after B. uniformis stimulation in the gut and TME. B. uniformis and other Bacteroides spp. can modulate immune responses via their unique capsule polysaccharides, but their impact on anti-tumor immunity is unknown. Therefore, I hypothesize that B. uniformis directly activates NK cells, through its capsule molecules, resulting in remodeling of the immune landscape in the tumor microenvironment, and enhancing anti- tumor immunity to protect against CRC. I will investigate this hypothesis along three lines of interrogation. Specific Aim 1: Identify the bacterial factor by which B. uniformis modulates NK cell activation and cytotoxicity. Specific Aim 2: Determine how strain level genetic variations in B. uniformis influences immune activation and CRC development. Specific Aim 3: Investigate how bacterial induced NK activation in the tumor microenvironment promotes anti-tumor immunity. By completion of this proposed work I will have a more nuanced understanding of the mechanisms by which B. uniformis prevents tumor formation via NK cells in the TME, which will lead to translational progress in innovative diagnostics and novel therapies for patients with CRC.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Exercise is an important modulator of human health, and mechanical loading during strength and resistance- based exercise is well known to positively impact bone mass by enhancing osteoblast-mediated bone formation. However, there is a fundamental gap in understanding the response of bone to aerobic metabolic demands found in activities like running and cycling, and how those demands interact with the mechanical loads placed on the skeleton during exercise to differentially modulate bone mass. Emerging evidence shows that aerobic exercise induces acute elevations in the bone resorption marker carboxy-terminal telopeptide of type I collagen (CTX) without compensatory changes in bone formation, suggesting an immediate and unexplained pro-resorptive response. While well-documented, there is substantial lack of prior rigor regarding the cell source, extent, and purpose of elevated bone resorption during aerobic exercise. These questions are important because they may help facilitate the development of more precise recommendations for aerobic exercise to modulate bone health. Furthermore, they may provide greater understanding of factors governing bone loss in conditions of high metabolic demands or low energy availability that often result in fractures or additional disability in vulnerable populations. The central hypotheses of this proposal are that the source of CTX elevation during exercise exists on a spectrum between osteocyte perilacunar remodeling (PLR) and osteoclast resorption, the extent of which is governed by energy demands during exercise, and that CTX elevation is proportional to amino acid flux into skeletal muscle. These hypotheses will be tested in three distinct, non-overlapping specific aims. Aim 1 will determine the source of elevated CTX during aerobic exercise. We will selectively inhibit osteocyte perilacunar remodeling using MMP13ocy-/- mice and compare exercise-induced elevations in CTX to mice treated with pamidronate, which we have previously shown can inhibit osteoclast activity in the face of sustained PLR, or cathepsin K inhibitor, which selectively inhibits collagen degradation, but not demineralization of the bone ECM. Aim 2 will determine the relationship between CTX elevation and energy demands during exercise. We will run well-trained human subjects, and rats bred for high and low aerobic capacity, at treadmill speeds correlating with varying oxidative and non-oxidative energy demands. Subjects will run on commercial (human) and custom (rat) treadmill-interface systems to uncouple metabolic demands from biomechanical loading, and we will further modulate energy demands in the presence or absence of carbohydrate supplementation. Lastly, Aim 3 will evaluate the relationship between CTX elevation and amino acid distribution to skeletal muscle during aerobic exercise. We will assay circulating and muscle-targeted amino acids in rodent samples to quantify amino acid flux to the muscle in relation to both modulation of bone turnover and energy demands of the exercise itself.

NCI - National Cancer Institute

Glioblastoma (GBM) is an aggressive brain tumor that recurs in treatment-refractory form within months of initial standard-of-care (SOC) treatment. Extensive study of primary (pre-treatment) GBM has revealed numerous characteristics that impede treatment, including marked intratumoral genetic, transcriptional, and spatial heterogeneity; rapid infiltration throughout the brain parenchyma; a uniquely compromised immune microenvironment; and immunosuppression beyond the central nervous system. However, the mechanisms by which these factors enable disease progression are not understood, and the critical disease entity -- recurrent GBM -- is comparatively understudied. The central hypothesis of this study is that identifying the unique characteristics of recurrent GBM and the mechanisms driving its evolution from primary GBM will reveal new therapeutic targets. Our overall objective is to identify transcriptional, genetic, neuronal, and immunological factors that influence GBM formation, progression, and treatment response, with an emphasis on therapeutically targetable ligand-receptor interactions. We utilize unique human tissue repositories, novel experimental designs, and spatially-resolved single-cell approaches with the rationale that reversible interactions between tumor cells and diverse nonmalignant cells drive tumor formation, progression, and drug resistance. We pursue our objective through three Specific Aims: (1) Identify unique molecular, cellular, and spatial features of recurrent human GBM; (2) Identify mechanisms underlying distal pathological effects of GBM, including distant brain infiltration and systemic immunosuppression, in whole-brain and whole-body autopsy settings; and (3) Elucidate the dynamic reconfiguration of the GBM ecosystem during tumor formation and treatment response using mouse models, single-cell lineage tracing, and spatial transcriptomics. In Aim 1, we will integrate spatial transcriptomics (ST) with novel long-read single-nucleus RNA-sequencing (snRNA-seq) and exome sequencing to identify cell states, single-cell clonal architecture, multicellular niches, and heterotypic ligand-receptor interactions specific to recurrent GBM. In Aim 2, we will leverage unique resources at MGH that enable the analysis of diverse brain regions and peripheral immune organs in post-mortem GBM patients. We will combine snRNA-seq, ST, and single-cell T-cell receptor sequencing to study brain invasion and immunosuppression within the brain and peripheral immune system. In Aim 3, we will perform time-resolved single-cell RNA-seq (scRNA-seq) and ST in a mouse model of GBM to map the temporal and spatial co-evolution of tumor and immune cells during tumor development and response to SOC. Critically, we will integrate single-cell lineage tracing with scRNA-seq and ST to distinguish among possible mechanisms by which treatment reconfigures the tumor ecosystem. This study addresses innovative questions using unique resources, novel study designs, and new combinations of genomic and computational approaches. The work is significant because it will enable the discovery of therapeutic vulnerabilities associated with GBM development, progression, and recurrence.

NIH

Custom accommodative insoles are prescribed to patients with diabetes who are at risk of developing plantar ulcers. Custom insoles aim to reduce plantar pressures through an accommodating insole with a weight-bearing surface that conforms to the patient’s foot. The current standard of care (SoC) insoles are typically constructed from an EVA foam block milled to match a patient’s foot shape, captured via a foam crush box, and then covered with a uniform sheet of poron and plastazote foam. The material properties of these insoles are generic and not patient specific. We have developed insoles that not only have patient specific shape but also patient specific regions of modulated stiffness. Our insoles are more effective in reducing plantar pressure in the areas of high pressure than the current SoC insoles in a healthy population and investigation is underway with similar preliminary results in individuals at risk of developing a plantar ulcer. Our work suggests that both foot shape and plantar pressure are important factors when designing accommodative insoles and offer the clinician a new tool to better customize the insole to the patient’s specific needs. Clinically, direct foot-shape scanners are already being implemented. However, it is critical to understand if these scanners distort the shape of the foot compared to the traditional foam crush box and how reliable the various scanning methods are within and across clinicians. Evaluations of intra-rater reliability of basic measures of foot shape (i.e., length and width) have been conducted, however, more comprehensive methods evaluating the plantar surface such as arch volume and overall surface differences are critical when designing an insole, given that the insole must conform to the foot shape. Thus, the purpose of this proposal is to evaluate the methods for obtaining shape and their effects on accommodative insole design and the ability to offload high-pressure areas in people with diabetes. Additionally, plantar pressure is not incorporated into the clinical design of SoC insoles. Compared to our current in-shoe pressure capture method, barefoot walking plantar pressure is a much more practical metric for clinicians to obtain. In this project, we will also determine if designing insoles with barefoot or in-shoe plantar pressure yields similar reductions in plantar pressure. Aim 1a: Quantify the inter- and intra-clinician reliability of three methods of capturing foot shape (crush box, Tiger scanner, structure sensor). Aim 1b: Quantify the differences in foot shape obtained using three methods of capturing foot shape. Aim 2: Evaluate the ability of insoles designed using the various foot scanning methods to offload areas of high pressure in the diabetic foot. Methods: Aims 1a and b: Three clinicians will take three shape measurements of the feet of ten healthy individuals with each of the following foot shape capture devices. Standard 1-D measurements and more comprehensive methods of evaluating the plantar surface will be calculated. Inter- and intra-clinician reliability will be calculated for each of the foot-shape capture methods (Aim 1a) and differences in the overall foot shape across devices compared (Aim 1b). Aim 2: 20 individuals with diabetes and elevated plantar pressures will undergo a clinical foot exam, have foot shape captured (using the three techniques described above), and have plantar pressures recorded during barefoot and shod walking. Seven sets of insoles will be manufactured for each participant, one pair of the SoC insole and six pairs of 3D printed insoles using the various foot shape and pressure capture methods. In-shoe plantar pressure will be measured during walking while wearing the insoles. Participants will also complete a comfort and usability survey after each insole condition. Peak pressure and pressure time integral in four regions (first metatarsal head, second metatarsal head, third to fifth metatarsal head, and offloading region) of the foot will be compared. VHA currently cares for 563,000 Veterans defined by the Prevention of Amputation in Veterans Everywhere (PAVE) Program as being at high risk for ulceration and amputation. Developing custom offloading insoles that are more efficacious than the current standard of care by leveraging advances in 3D printing has the potential to reduce rates of amputation and morbidity in our Veteran population.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: Protein targeting is essential for maintaining cellular organization and function across the tree of life. Signal Recognition Particle (SRP) is a ribonucleoprotein complex that plays an important role in this process, co- translationally targeting 30% of the proteome for membrane insertion or secretion. SRP recognizes the signal sequence of secretory proteins, pauses translation, and guides the ribosome-nascent chain complex to the endoplasmic reticulum. Errors in SRP-dependent protein targeting lead to protein aggregation and fitness defects, and are associated with developmental disorders, neurodegeneration, and cancer. Despite SRP’s important function, the mechanisms underlying its assembly remain poorly understood. Specifically, the factors involved in SRP assembly and how assembly is subcellularly coordinated remains largely unknown. Therefore, the goal of this proposal is to identify SRP assembly factors and characterize their mechanism in human cells. Structurally, SRP consists of six proteins and a 300-nucleotide noncoding RNA, 7SL. Using microscopy, we and others have observed that several of these components localize to nucleoli, which are biomolecular condensates that form through liquid-liquid phase separation and facilitate ribosome biogenesis. Based on these observations, I hypothesize that nucleoli concentrate factors that mediate 7SL maturation, which is necessary for SRP assembly. This will be tested through two specific aims. In Aim 1, I will identify 7SL RNA-binding proteins and investigate their role in SRP assembly and 7SL folding. In Aim 2, I will employ CRISPR screening to identify nucleolar SRP assembly factors and determine whether their condensation contributes to function. By testing whether nucleoli coordinate SRP assembly, this work will advance our understanding of biomolecular condensates and ribonucleoprotein biogenesis, revealing novel therapeutic targets for diseases that arise from protein mistargeting. Furthermore, this study constitutes the research arm of a comprehensive training plan that facilitates my development as a physician-scientist. Throughout this proposal, I will have mentored training in molecular biology research and gain the skills necessary to become an independent investigator, including experimental design, scientific writing, and presentation skills. This work will be conducted at the Omenn-Darling Bioengineering Institute at Princeton University, which has state-of-the-art microscopy, flow cytometry, and genomics equipment. My research training will be complemented with longitudinal clinical training in precision medicine and hematology-oncology at the Rutgers Cancer Institute. Together, my research and clinical training will provide a strong foundation for pursuing a career as an independent physician-scientist.

NIDCR - National Institute of Dental and Craniofacial Research

Project Summary Metabolic Syndrome (MetS) affects over one-third of U.S. adults and is closely linked with oral health issues, particularly periodontitis. The bidirectional relationship between MetS and periodontitis exacerbates both conditions, but the underlying mechanisms remain unclear. Our preliminary R03 data indicate that MetS alters the oral microbiome, creating an ‘at-risk-for-harm’ environment even in the absence of clinical periodontal disease. This project aims to elucidate these mechanisms through a longitudinal systems biology approach, focusing on how restoring metabolic health impacts the functional oral ecosystem. We hypothesize that improved metabolic health will shift the oral microbiome towards a eubiotic state, reduce pathogenic strains, and alter the metabolome to a less inflammatory profile. We test our hypothesis in the following specific aims. In Specific Aim 1: We will track the oral microbiome's temporal dynamics in MetS and Metabolically Healthy Obese (MHO) cohorts undergoing lifestyle or surgical interventions, comparing them with healthy and periodontal disease cohorts. In Specific Aim 2: We will analyze changes in the oral metabolome post-metabolic intervention using advanced mass spectrometry techniques. In Specific Aim 3: We aim to identify and validate oral biomarkers and risk indicators linked to metabolic health improvements. This study will use a causal mediation framework to explore direct and indirect effects, providing crucial insights into the MetS-periodontal health relationship and aiding in the development of prognostic biomarkers and risk indicators in the future.

National Park Service

United State Department of the Interior, National Park Service (NPS) Notice of Intent to Award. This is not a request for applications. This funding opportunity is to provide public notice of NPS's intention to fund the following project activities without full and open competition. Task Agreement # P15AC00255 under Cooperative Agreement P11AC8R001 with the University of Nevada, Reno, a partner under the Californian Cooperative Ecosystem Studies Unit, for project titled "Determining the Influence of a Predaceous Diving Beetle (Neoclypeodytes cinctellus) on the Devils Hole pupfish (Cyprinodon diabolis) and its Prey"

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Whether due to injury, disease, or as a consequence of cancer chemotherapy, millions of Americans, both young and old, experience sensory neuropathy. This condition, characterized by numbness, tingling, and disruptions to gait, degrades the quality of life. The sheer diversity of cells that underpin the mechanical senses of touch, hearing, and proprioception pose a barrier to understanding their function in health and their dysfunction in disease. Rapid mechanosensation responsible for low-threshold touch sensation depends on the activation of specialized mechanosensitive (MS) ion channels. The proteins forming MS channels in humans and other animals have come to light over the past two decades, yet how mechanical energy causes their activation remains poorly understood. Some MS channels seem to respond to changes in membrane tension, others depend on coupling to the cytoskeleton, and still others require coupling to extracellular matrix (ECM) structures. Whereas a subset of neuropathies arise from faulty nerve transmission, many other forms involve defects in coupling MS channels to sensory stimulation or from defects in the channels themselves. This project seeks to understand how sensory stimuli is coupled to MS channel activation in vivo. Very few biological models are amenable to in vivo neuronal biophysics, including voltage-clamp of sensory neurons and to imaging MS channel complexes in living animals. The model organism C. elegans fulfills these requirements. In this research we will combine established techniques for in vivo whole-cell patch-clamp electrophysiology with cutting-edge, genetically encoded tools to visualize and measure touch-induced mechanical strain in touch receptor neurons. A large toolbox of existing mutations affecting the cytoskeleton and ECM will be used to perturb the coupling between sensory stimulation and MS channel activation, delineating the role played by each structure in this fundamental sensory process. Ample evidence indicates that the mechanics of touch are conserved even among distantly related animals, suggesting that what is learned through this research training plan has the potential to provide insight into the factors that give rise to sensory neuropathy and to other mechanosensory abnormalities.

NIMH - National Institute of Mental Health

PROJECT SUMMARY/ABSTRACT Excess dopamine in the dorsal striatum is thought to be a key driver of schizophrenia. Accordingly, there is a strong corre- lation between the potency of antipsychotic drugs and their affinity for D2 dopamine receptors (D2Rs). However, tradi- tional D2R-binding drugs are not effective for psychosis in ~30% of patients, do not address cognitive and negative symp- toms, and have many adverse effects. Recently, several new therapies have emerged that do not interact with D2Rs. These include promising drugs like xanomeline, an agonist of M1 and M4 acetylcholine receptors. One promise of this new drug class is its potential to work for more patients and address more symptoms than traditional antipsychotics. Capitalizing on this novel therapeutic mechanism requires a deeper understanding of how the drug class works. Recently, our lab used in vivo imaging to determine how two of these drugs (xanomeline and the M4 receptor selective modulator VU0467154) compare to more traditional antipsychotics. Paradoxically, we found that clinical antipsychotic efficacy was more strongly associated with the normalization of activity in striatal neurons that express D1 rather than D2 dopamine receptors (Yun et al., 2023). This was true for both traditional antipsychotics and the newer cholinergic receptor agonists—suggesting the two drug classes may have partly overlapping end effects in the brain. In parallel to these imaging studies, we developed two approaches to selectively activate (either transiently or persistently) dopamine projections to the dorsal striatum. Both approaches affect behavioral processes related to the symptoms of schizophrenia and are compatible with in vivo record- ing. Here will combine these models with in vivo imaging or microdialysis to understand the mechanism-of-action of novel cholinergic antipsychotics and use behavioral testing to explore the range of symptoms for which they may be ef- fective. Specifically, we will use dual-color in vivo imaging to simultaneously record striatal acetylcholine transmission and calcium activity in D1 or D2 receptor-expressing spiny-projection neurons (SPNs). After determining how acetylcho- line relates to D1-/D2-SPN activity and how each relates to locomotor activity, we will activate nigrostriatal dopamine neurons and observe the effects on acetylcholine and D1-/D2-SPN activity. After determining these effects, we will ask how different cholinergic receptor agonists or modulators (xanomeline, M1 agonist, M4 agonist, or an M1 positive allo- steric modulator) affect the relationship between acetylcholine and D1-/D2-SPN activity under normal and hyperdopamin- ergic conditions. The drugs we will test will allow us to directly compare receptor-specific agonism and positive allosteric modulation in isolation or in combination. Next, we will use the same drugs with our animal model of persistent nigrostri- atal dopamine neuron activation. We will use in vivo microdialysis to determine how striatal neurochemistry is altered in these animals and how different cholinergic drugs affect these changes. Finally, we will use behavior (working memory, social exploration, and auditory perception) to determine whether different cholinergic drug types normalize different be- havioral processes in this animal model. Altogether we expect these experiments will advance this new therapeutic strat- egy for psychosis by elucidating the mechanistic basis for its effects on striatal activity, neurochemistry, and behavior. In doing so, we hope to catalyze the development of better and more comprehensive treatments for psychotic disorders.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT Neuroplasticity is a key feature of cortical systems allowing for flexible shaping of pyramidal cell activity necessary for learning, adaptation, and recovery from injury. A shared feature of neuroplasticity across cortical and subcortical regions is the involvement of inhibitory interneurons in refining pyramidal cell activity. In the motor cortex, pyramidal cells tile their activity to skilled behaviors, likely being shaped by local inhibitory interneurons. However, how pyramidal cell sequencing emerges in the motor cortex to produce and adapt skilled behaviors remains unknown. Delineating the mechanisms and circuitry involved in shaping pyramidal cell sequencing is crucial to understanding the fundamental rules of motor plasticity, and to inform future therapeutic interventions to enhance neuroplasticity. While previous studies have investigated the role of interneuron subtypes in motor plasticity; the predominant interneuron in layer 1 of cortex, neuron-derived neurotrophic factor (NDNF) neurons, has yet to be researched. NDNF neurons have been characterized across cortical regions including auditory, visual, somatosensory, and prefrontal cortex. While their functional role across these cortical regions continues to be explored, there is evidence these neurons regulate pyramidal cell inputs, attenuating top-down inputs and gain-controlling bottom-up inputs. Skilled behavior learning and execution involves the dynamic combination of top-down inputs originating from the basal ganglia and bottom-up inputs deriving from the cerebellum to shape pyramidal cell activity. Our overall hypothesis in this proposal is that motor learning and execution rely on NDNF neurons to refine pyramidal cell activity through regulation of top-down and bottom-up inputs. To test this, I will first determine the anatomical localization of NDNF neuron somata, dendrites, and axons. Then, I will establish the dynamics of NDNF neurons in M1 during motor execution and learning using two-photon calcium imaging. Lastly, I will casually test the role of NDNF neurons on shaping pyramidal cell activity by optogenetically activating and suppressing NDNF neurons while performing large-scale neural recording of pyramidal cells. In all, this work will establish the role of motor cortex NDNF neurons in neuroplasticity.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT Bacteria Microcompartments (BMCs) are pseudo-organelles comprised of a highly organized, semi-permeable protein shell which self-assembles around components of specific metabolic pathways. BMCs offer bacteria an advantage to survive in harsh conditions, to organize the cytosol by separating reactive components from each other, or to increase efficiency of enzymatic pathways by tuning local concentrations. Genetic models suggest that the compact organization of the BMC loci is ideally suited for lateral transfer to other bacteria, increasing the potential for greater prevalence of this feature in the future. Understanding the mechanisms of BMC assembly, organization, and stability are important to developing new approaches involving human-bacteria interactions and symbiosis. The model BMC is a key component in the CO2 concentrating mechanism (CCM) found in cyanobacteria called the carboxysome (CB). CBs sequester Rubisco with carbonic anhydrase and CO2 increasing carbon fixing efficiency. Rubisco is the most abundant enzyme on earth and the protein responsible for carbon fixation in plants, bacteria, and algae. Rubisco has low selectivity for its substrate CO2 and has a slow enzymatic rate, resulting in an inefficient metabolic pathway. By sequestering Rubisco in CBs along with additional co-factors, cyanobacteria and many chemoautotrophs have enhanced carbon fixation. We propose studying shell assembly and cargo organization in vivo and in vitro using cryogenic electron tomography (cryoET) and live cell imaging using total internal reflection fluorescence microscopy (TIRFM) to understand internal and external factors that affect BMC stability, permeability, and function. Understanding how this model BMC organizes during assembly and throughout its life cycle will be critical for developing potential bioreactors with custom cargo for pharmaceutical applications or increasing carbon fixation in photosynthetic organizations for improved crop yields and decreasing atmospheric carbon.

NIMH - National Institute of Mental Health

Project Summary Neuromodulators like dopamine are crucial for learning, driving synaptic changes in neurons, and shaping behavior. Furthermore, dysfunction in dopamine signaling is a hallmark of almost all psychiatric disorders. Traditionally, dopamine was thought to broadcast diffuse signals across broad brain regions. However, emerging evidence points to a more targeted mechanism, where dopamine acts on specific neurons engaged in behaviorally relevant tasks. This proposal raises a critical question: How does a widely distributed neuromodulator like dopamine contribute to precise, neuron-specific learning? This project will explore how dopamine shapes learning in sparse cortical populations. We will use two-photon microscopy and genetically encoded sensors to simultaneously track dopamine release and neuronal activity in real-time as mice perform a neuroprosthetic control task. We will use this task because it allows us to directly identify the neurons that drive behavior, providing a unique opportunity to examine whether dopamine signals are more focused on task-relevant neurons. We will measure both dopamine release and the activity of specific neurons involved in behavior to determine whether dopamine shapes learning at a single neuron level or across broader networks, and what type of information can be conveyed by dopamine signals at different levels of spatial resolution. This study will help us better understand the brain mechanisms underlying learning by revealing fundamental mechanisms of how dopamine modulates neural circuits. Our results will help us understand how disruptions in these processes contribute to conditions like depression, schizophrenia, and addiction, which involve pathological learning. This research could lead to more targeted therapies focusing on dopamine-related brain circuits, ultimately offering better treatment options for people with cognitive dysfunction and maladaptive behavioral patterns.

NIAID - National Institute of Allergy and Infectious Diseases

The World Health Organization recommends that all children be vaccinated against rotavirus, the most common cause of severe childhood diarrhea and a major cause of morbidity and mortality worldwide. Two oral rotavirus vaccines have proven to be highly efficacious against childhood diarrhea in high- and middle-income countries (85%-98%). However, virtually all trials in sub-Saharan Africa and south Asia have shown lower immune response and much lower vaccine efficacy (40%-64%), leaving many children unprotected. The ultimate goal of this research is to enhance the efficacy of oral vaccines in infants in low-resource settings by identifying the biologic basis for their underperformance in these settings. The research team hypothesizes that the predominant determinants of vaccine failure in low-resource settings are micronutrient deficiencies. Micronutrients, particularly iron, zinc, vitamin A, and vitamin D, play an important role in maintaining the integrity of gut mucosal immunity, a primary site of interaction for oral vaccines, and are critical in activating T and B-cell responses that are essential to oral vaccine response. As a result, key micronutrient deficiencies among mothers in pregnancy may result in a nutrient-deficient fetal environment that handicaps development of a functional infant immune system, while deficiency in infants may also compromise immune development in early live, leaving them unable to mount an effective oral vaccine response. This study will evaluate the associations of four key micronutrients (iron, zinc, vitamin A, and vitamin D) in 250 mother-infant pairs with infants’ failure to develop an effective immune response using plasma samples from a completed randomized, placebo-controlled vaccine efficacy trial in Niger. These four micronutrients were selected for the robust scientific evidence for their critical role in pathways key to oral vaccine efficacy (mucosal immunity and immune cell development) and the relevance to low-resource settings, where deficiency is common. In this study, we aim to test two hypotheses: Aim 1: Low maternal micronutrient concentrations are associated with impaired infant immune response to oral rotavirus vaccine (anti-rotavirus IgA seroconversion at 28 days post-Dose 3). Aim 2: Low micronutrient concentrations in infants are associated with impaired immune response to oral rotavirus vaccine. This study will guide development of new randomized clinical trials (R01) that test the causal effect of micronutrient supplementation on rotavirus vaccine performance. Implementation of such interventions at scale will boost the efficacy of oral vaccines that have historically performed poorly, increasing the impact of immunization programs worldwide.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT The brain consumes nearly 20% of the body’s ATP despite accounting for only 2% of body weight, highlighting its disproportionate energy demand. As the cells of the brain also exhibit a limited regenerative capacity, sustained bioenergetic failure often results in a permanent loss of cognitive, sensorimotor, and autonomic function. While neurodegenerative and neurodevelopmental conditions are often multifactorial, one common theme observed across these diseases is altered metabolic homeostasis. To meet the tremendous energetic demands of intercellular communication, neurons rely on highly efficient ATP production via mitochondrial oxidative phosphorylation (OxPhos). In addition to ATP, OxPhos yields reactive oxygen species (ROS) shown to oxidize cellular lipids, proteins, and nucleic acids. While decades of research have investigated the consequences of ROS accumulation in various pathologies and aging, the role of ROS in healthy neuronal function is incompletely understood. Membrane-diffusible mitochondrial hydrogen peroxide (mtH2O2), a specific form of ROS, has become the focus of recent research due to its reported interactions with redox modifiable cysteine residues – coined redox switches. As mtH2O2 is produced in the mitochondrial matrix in a metabolic performance dependent manner, it likely exhibits direct influence on local mitochondrial dynamics to shape metabolism and maintain a healthy mitochondrial network. Our preliminary data in primary mouse cortical neurons suggests that mtH2O2 serves a regulatory role in mitochondrial fission/fusion balance. Following attenuation of mtH2O2 via depletion of superoxide dismutase 2 (SOD2), we observed a reduction in mitochondrial size within neuronal dendrites. To ascertain if this change in size is a result of impaired mitochondrial fission/fusion balance, we performed time lapse fluorescent microscopy on live primary neurons and found that while SOD2 ablation decreases the frequency of both fission and fusion events, it preferentially inhibits fusion. Thus, we hypothesize that mtH2O2 dictates local neuronal mitochondrial dynamics. We have devised two independent aims to test this hypothesis. In the first aim, we will determine the compartment- specific signaling capacity of mtH2O2 by visualizing H2O2 spatial abundance in the mitochondrial matrix and at the outer mitochondrial membrane in neuronal dendrites, somas and axons using a novel genetically encoded H2O2 reporter, HyPer7. In the second aim, we will quantify changes in mitochondrial fission and fusion following genetic manipulation of mitochondrial antioxidants – resulting in physiologically relevant changes in mtH2O2 levels – then employ targeted proteomics to identify differences in the oxidation profiles of known fission and fusion proteins. If successful, our work will characterize an essential role of mtH2O2 in mitochondrial health by its direct regulation of proteins involved in mitochondrial remodeling.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Poly (ADP-ribose) polymerase inhibitors (PARPi) are synthetically lethal in cells with homologous recombination (HR) defects, a phenotype of certain cancers. BRCA1 is one of the most commonly mutated genes in hereditary, HR-deficient cancers. Unfortunately, patients with HR-deficient cancers commonly acquire resistance to PARPi, and the mechanisms of PARPi-induced cytotoxicity are underexplored. Understanding these pathways could provide insight into how patients acquire PARPi resistance. The PARPi cytotoxic response requires cells to transit mitosis, and cells that persist through PARPi treatment (persister cells) arrest from the cell cycle. Thus, determining this mechanism of cell cycle commitment is central to understanding PARPi-induced cytotoxicity as cells face two potential fates in response to PARPi: undergo mitosis and trigger cell death or arrest from the cell cycle and persist. My preliminary data suggests that arrested persister cells have decreased levels of Lamin B1 (LMNB1, a nuclear lamina protein): loss of which is necessary and sufficient to induce cell cycle arrest in other contexts. Thus, LMNB1 downregulation may contribute to the cell cycle arrest mechanism in these persister cells. For PARPi-treated cells that undergo mitosis-dependent cell death, the pathways governing this cytotoxicity are unclear. PARPi have been shown to synergize with other drugs to induce various forms of regulated cell death (RCD) in different cell lines, suggesting that multiple forms of RCD could be triggered in BRCA1-deficient cells. A systematic investigation is needed to determine the RCD mechanisms involved in the PARPi response and the upstream regulatory pathways that initiate them. One hypothesis of a contributing regulatory pathway is cyclic GMP-AMP synthase (cGAS) / stimulator of interferon genes (STING) signaling, which canonically induces innate immune signaling in response to cytosolic DNA. cGAS/STING signaling is increased in HR-deficient, PARPi-treated cells. Higher instances of mitotic errors, including persistent DNA bridges, are also observed in response to PARPi treatment. As these structures are prone to rupture, leading to loss of nuclear envelope integrity, we hypothesize that they could be surveilled by cGAS to signal RCD in the PARPi response. The objective of this proposal is to determine the mechanisms that confer vulnerability of BRCA1-deficient cells to PARPi-induced cytotoxicity. Using BRCA1-deficient breast and ovarian cancer cell lines as models, in Aim 1 I will determine mechanism of cell cycle evasion that allows cells to persist through PARPi treatment, specifically investigating LMNB1 downregulation as a contributing pathway. In Aim 2 I will conduct an arrayed CRISPR screen targeting RCD driver genes and other regulatory pathways to determine the mechanisms of PARPi-induced cytotoxicity in cycling cells, including investigating a role for cGAS/STING signaling. Overall, this proposal will enhance our understanding of how cells evade or succumb to PARPi-induced cell death. This work could inform combination therapies with PARPi to combat PARPi resistance in patients with BRCA1-deficient cancers.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Cranial neural crest cells are a population of multipotent stem cells essential for craniofacial development that contribute to the sensory ganglia and craniofacial skeleton. Neural crest cells are specified during neurulation before undergoing an epithelial-to-mesenchymal transition (EMT) and migration; while we appreciate the complex network of signaling pathways and transcription factors underlying specification and migration, the regulation of these events remains incompletely understood. We have recently identified that expression of the gene encoding Raftlin-2 is upregulated in neural crest cells during specification and maintained throughout migration. Raftlin proteins localize to the plasma membrane where they are reported to organize lipid raft domains and regulate diverse cell signaling events, leading us to hypothesize that Raftlin-2 contributes to the development of neural crest cells through an impact on cell signaling. In preliminary studies probing Raftlin-2 function in the cranial neural crest of avian embryos using multiple knockdown strategies, we observed reduced expression of several genes required for neural crest specification, suggesting that Raftlin-2 is essential for the establishment of the neural crest domain. Aim 1 will investigate the role of Raftlin-2 in regulating specification by using immunohistochemistry to measure neural crest specifier genes expression. Since specifier gene expression is initiated by cell signaling pathways including Wnts and Bone Morphogenetic Proteins, I will next use signaling activity-sensitive reporter constructs, and fluorescent in situ hybridization to investigate if Raftlin-2 regulates the activity of these pathways to govern specification. Finally, I will use RNA sequencing in Raftlin-2 deficient cranial neural crest cells to determine additional targets and signaling pathways dependent on Raftlin- 2 function. Our preliminary results also showed reduced neural crest migration area after Raftlin-2 knockdown compared to controls in vivo, and we find that Raftlin-2 loss of function resulted in decreased migratory persistence ex vivo, which led us to hypothesize that Raftlin-2 is necessary for effective neural crest migration and adhesion after specification. Aim 2 will test this hypothesis using ex vivo explants to assay neural crest cell directional migration and focal adhesion dynamics during chemotaxis. I will also use quantitative western blot analysis to probe the activation states of molecular effector proteins downstream of chemotactic signals to define and subsequently test the precise mechanisms of Raftlin-2 function during migration. Together these experiments will uncover how Raftlin-2 impacts developmental signaling pathways necessary for cranial neural crest cell specification and migration. These results will advance our understanding of how membrane organization regulates craniofacial development insight and will provide novel insights that can be used to treat craniofacial anomalies.

NSF

NON-TECHNICAL SUMMARY: DNA is the molecule in your cells that stores your genetic information, but it is also an important building material on the nanoscale. The base-pairing rules of DNA—A binds to T, C binds to G—allow engineers to easily assemble different shapes and structures. However, the simplicity of DNA also limits its functionality. By adding particular molecules called condensing agents to DNA, our goal is to bend, loop, stack, or reconfigure DNA on command. This reconfiguring of the DNA could impart dramatic properties to the DNA material, including decreased size, increased stability, increased stiffness, decreased permeability, or increased electrical conductivity. We will study three questions: How do condensing agents bend and loop DNA? How do condensing agents reconfigure DNA? And, how can we use condensing agents to engineer particular DNA materials? Answers to these questions will impact our use of DNA materials in various applications, including immunotherapies, carbon capture technology, tissue engineering, biosensing, or nanorobotics. More broadly, the science in the grant will be driven entirely by undergraduate researchers, presenting an incredible educational opportunity to train the next generation of scientists and engineers. At the same time, the training modules created by the undergraduates will then be used to train college/university instructors in a nationwide program. TECHNICAL SUMMARY: DNA is a ubiquitous biomaterial that is easy to engineer. However, this simplicity limits functional versatility. Here, we will use a biomimetic approach and couple DNA to the condensing agents, which typically fold, loop, condense, or reconfigure the DNA inside the cell. Condensing agents are a class of positively charged molecules that could be used to self-assemble and reconfigure DNA materials on cue, creating increased condensation, increased stability, increased stiffness, decreased permeability, or increased electrical conductivity. Our goal is to determine the rules for how condensing agents bend, loop, condense, and reconfigure DNA molecules and DNA assemblies. While researchers have been studying how condensing agents condense DNA for 40 years, the answers still remain elusive. We will use new single molecule techniques, as well as a “bottom-up” approach, starting with short DNA molecules and working up to larger DNA assemblies, to study the structures, pathways, mechanics, and energetics of the process. In addition, we will—for the first time—look at how condensing agents reconfigure DNA molecules with several assembled nucleosomes. This project will pave the way for DNA biomaterials with exquisite regulatory, electrical, mechanical, or geometric control. This work will be driven entirely by undergraduate researchers, presenting an incredible educational opportunity to train the next generation of scientists and engineers. Training modules created by the undergraduates will then be used to train college/university instructors in a nationwide program. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NINDS - National Institute of Neurological Disorders and Stroke

The long-term goal of this project is to understand how loss of progranulin (PGRN) causes neurodegeneration in frontotemporal degeneration (FTD) and neuronal ceroid lipofuscinosis (NCL), commonly called Batten’s disease. FTD and NCL are fatal and currently incurable. FTD and NCL have distinct clinical manifestations but share a causative genetic factor, the GRN gene, which encodes PGRN. PGRN is an 80 kDa secreted glycoprotein composed of 7.5 tandem proteins called granulins. Heterozygous GRN mutations decrease PGRN levels and are a common cause of FTD, the most frequent dementia before the age of 60. Importantly, complete loss of PGRN through homozygous GRN mutations causes the neurodegenerative lysosome storage disease NCL type 11 (CLN11). Thus, PGRN activity is important for lysosome function and brain health, yet the function of PGRN is unclear. Our lab discovered that PGRN localizes to the lysosome, is critical for lysosome homeostasis, and is rapidly processed in the lysosome into 7 individual stable 6-kDa granulins. New preliminary data show that treatment of Grn-/- mice with a single granulin, granulin-4, fully ameliorates neuropathology, lysosome dysfunction, and neuroinflammation. These data lead us to hypothesize that a novel receptor facilities lysosomal trafficking of PGRN and processing into granulins, which function to maintain bis(monoacylglycerol)phosphate (BMP) lipid levels that are necessary for lysosomal lipid catabolism to prevent neurodegeneration. Our team has the expertise and necessary tools to bring clarity to the function of granulins and test this novel hypothesis in three aims. In Aim 1, we will test the ability of each granulin to ameliorate lysosome dysfunction and neurodegeneration. In Aim 2, we will delineate the molecular pathways that traffic PGRN to the lysosome to produce granulins. In Aim 3, we will dissect the molecular mechanisms of granulin function in lysosome lipid metabolism. Completion of these studies will rigorously test the novel hypothesis that lysosomal granulins are the bioactive functional products of PGRN and potential pre- clinical therapeutics for FTD and NCL. Our results will provide clarity into the function of granulins in the lysosome, which has been a key question holding back the field. This project will help uncover how decreased PGRN and granulins cause lysosome dysfunction and neurodegeneration as well as elucidate new therapeutic targets for diseases caused by PGRN deficiency.